Methamphetamine induces autophagy as a pro-survival response against apoptotic endothelial cell death through the Kappa opioid receptor

Ma, J; Wan, J; Meng, J; Banerjee, S; Ramakrishnan, S; Roy, S

In conclusion, our data indicate that autophagy is an early response to the METH-induced stress and may have a protective role during early exposure to the drug. However, chronic exposure to METH allows for progression through autophagic flux and commits cells to apoptotic death. These results help to explain how chronic exposure of METH can contribute to endothelial injury. These findings clarify the link between autophagy and apoptosis signaling induced by METH abuse. Western blot. Cell lysates were prepared in RIPA buffer (Sigma) and stored at À 80 1C. Total protein concentration was determined by Bradford method. Wholecell extracts (20 mg) were electrophoresed on a sodium dodecyl sulfate/ polyacrylamide gel (SDS-PAGE), and electrotransferred to a nitrocellulose membranes (Amersham Biosciences, Pittsburgh, PA, USA). Blots were blocked by Tris-buffered saline solution containing 5% dry milk. Primary and secondary antibody-HRP conjugates were prepared in blocking buffer. Bands were visualized by the enhanced chemiluminescence (ECL) detection system (Amersham Biosciences). To verify the quality of loading, the blots were reprobed with antia-tubulin antibody (Santa Cruz Biotechnology).